Biomedical subjects

Dhundy R Bastola

Publications and source records attributed to Dhundy R Bastola.

8 recordsLinked to original sources

PubMed2006

Link test--A statistical method for finding prostate cancer biomarkers.

We present a new method, link-test, to select prostate cancer biomarkers from SELDI mass spectrometry and microarray data sets. Biomarkers selected by link-test are supported by data sets from both mRNA and protein levels, and therefore results in improved robustness. Link-test determines the level of significance of the association between a microarray marker and a specific mass spectrum marker by constructing background mass spectra distributions estimated by all human protein sequences in the SWISS-PROT database. The data set consist of both microarray and mass spectrometry data from prostate cancer patients and healthy controls. A list of statistically justified prostate cancer biomarkers is reported by link-test. Cross-validation results show high prediction accuracy using the identified biomarker panel. We also employ a text-mining approach with OMIM database to validate the cancer biomarkers. The study with link-test represents one of the first cross-platform studies of cancer biomarkers.

Algorithms↗

PubMed2005

Computational approach involving use of the internal transcribed spacer 1 region for identification of Mycobacterium species.

The rapid and reliable identification of clinically significant Mycobacterium species is a challenge for diagnostic laboratories. This study evaluates a unique sequence-dependent identification algorithm called MycoAlign for the differential identification of Mycobacterium species. The MycoAlign system uses pan-Mycobacterium-specific primer amplification in combination with a customized database and algorithm. The results of testing were compared with conventional phenotypic assays and GenBank sequence comparisons using the 16S rRNA target. Discrepant results were retested and evaluated using a third independent database. The custom database was generated using the hypervariable sequences of the internal transcribed spacer 1 (ITS-1) region of the rRNA gene complex from characterized Mycobacterium species. An automated sequence-validation process was used to control quality and specificity of evaluated sequence. A total of 181 Mycobacterium strains (22 reference strains and 159 phenotypically identified clinical isolates) and seven nonmycobacterial clinical isolates were evaluated in a comparative study to validate the accuracy of the MycoAlign algorithm. MycoAlign correctly identified all referenced strains and matched species in 94% of the phenotypically identified Mycobacterium clinical isolates. The ITS-1 sequence target showed a higher degree of specificity in terms of Mycobacterium identification than the 16S rRNA sequence by use of GenBank BLAST. This study showed the MycoAlign algorithm to be a reliable and rapid approach for the identification of Mycobacterium species and confirmed the superiority of the ITS-1 region sequence over the 16S rRNA gene sequence as a target for sequence-based species identification.

Base Sequence↗

PubMed2005

Mucin biosynthesis: upregulation of core 2 beta 1,6 N-acetylglucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line.

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan beta1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor of RA receptor alpha, implicating RA receptor alpha in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.

Cell Line↗

PubMed2004

Utilization of the relative complexity measure to construct a phylogenetic tree for fungi.

The relative complexity measure (RCM) is a new approach to evaluate relatedness of DNA sequences which eliminates the requirement to align sequences prior to analysis, a step required with standard reference methods. The value of the RCM approach to generate distance matrices for use in phylogenetic analysis of organisms has not been determined. This study compared RCM with the algorithmic and tree searching reference methods for phylogenetic analysis using fungal sequences. Sequences of the cytochrome b gene and the 18S rDNA gene were obtained from the GenBank database to determine feasibility of this method for phylogenetic relatedness. The RCM approach was also used to construct a phylogenetic tree using internal transcribed spacer (ITS) sequences from 23 medically relevant fungal species. The robustness of the RCM and reference approaches was determined by comparing the topology of seven medically relevant fungi within the phylogenetic trees generated after progressive removal of 10, 20, 30, 40 and 50% of the nucleotide bases from either the 5' or 3' end of the three genomic target sequences. The results demonstrated that the RCM method was equivalent to the reference methods for construction of phylogenetic trees from cytochrome b and 18S rDNA gene sequences. The phylogenetic tree constructed using the ITS sequence generated no contradictory topology. The RCM generated trees retained the appropriate topology after removal of up to 50% of the cytochrome b sequence, 40% of the ITS sequence, and 30% of the 18S gene target sequence. Comparatively, the reference methods failed to maintain topology after only a 10% sequence deletion for each genomic target. The results showed the RCM to be a reliable and robust computational approach for use in the construction of fungal phylogenetic trees without the requirement for prior sequence alignment.

Algorithms↗

PubMed2004

Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography.

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.

Animals↗

PubMed2003

Mucin biosynthesis: epidermal growth factor downregulates core 2 enzymes in a human airway adenocarcinoma cell line.

Enzymes which exhibit core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 beta1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I beta1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor alpha mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras-mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras-mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.

Adenocarcinoma↗

PubMed2002

Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking.

We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.

Active Transport, Cell Nucleus↗

PubMed2002

Downregulation of PTEN/MMAC/TEP1 expression in human prostate cancer cell line DU145 by growth stimuli.

Genetic alterations and/or deletion of the tumor suppressor gene PTEN/MMAC/TEP1 occur in many types of human cancer including prostate cancer. We describe the production of monoclonal antibody against recombinant human PTEN and the study of PTEN gene and protein expression in three commercially available human prostate cancer cell lines, PC-3, LNCaP, and DU 145. Northern blotting analyses showed that LNCaP and DU145 but not PC-3 cells expressed PTEN mRNA. However, Western blotting analyses using a monoclonal antibody against PTEN demonstrated the expression of PTEN protein in DU145 but not LNCaP cells. In DU 145 cells, PTEN expression at both the mRNA and protein levels inversely correlated with serum concentrations and levels of PKB/Akt phosphorylation. In addition, the basal activity of PKB/Akt as indicated by level of phosphorylation was higher in prostate cancer cells which do not express PTEN than that in the cells expressing wild type PTEN. Thus, PTEN may play a critical role in regulating cellular signaling in prostate cancer cells.

Antibodies, Monoclonal↗